Assay apparatuses, methods and reagents

ABSTRACT

Apparatuses, systems, method, reagents, and kits for conducting assays as well as process for their preparation are described. They are particularly well suited for conducting automated analysis in a multi-well plate assay format.

CROSS REFERENCE TO RELATED APPLICATIONS

The present patent application claims priority under 35 U.S.C. § 120 asa continuation application to U.S. non-provisional patent applicationSer. No. 14/147,216 filed on Jan. 3, 2014, which claims priority under35 U.S.C. § 119 (e) to U.S. provisional application No. 61/749,097entitled “Assay Apparatus, Methods and Reagents” filed on 4 Jan. 2013.The disclosures of the parent applications are incorporated by referencein their entireties. Reference is also made to U.S. ApplicationPublication Nos. 2011/0143947, 2012/0195800, 2007/0231217, 2009/0263904,and 2011/025663. The disclosures of each of these applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to apparatuses, systems, methods, reagents, andkits for conducting assays. Certain embodiments of the apparatuses,systems, methods, reagents, and kits of the invention may be used forconducting automated sampling, sample preparation, and/or sampleanalysis in a multi-well plate assay format.

BACKGROUND OF THE INVENTION

Numerous methods and systems have been developed for conductingchemical, biochemical, and/or biological assays. These methods andsystems are essential in a variety of applications including medicaldiagnostics, food and beverage testing, environmental monitoring,manufacturing quality control, drug discovery, and basic scientificresearch.

Multi-well assay plates (also known as microtiter plates or microplates)have become a standard format for processing and analysis of multiplesamples. Multi-well assay plates can take a variety of forms, sizes, andshapes. For convenience, some standards have appeared forinstrumentation used to process samples for high-throughput assays.Multi-well assay plates typically are made in standard sizes and shapes,and have standard arrangements of wells. Arrangements of wells includethose found in 96-well plates (12×8 array of wells), 384-well plates(24×16 array of wells), and 1536-well plates (48×32 array of wells). TheSociety for Biomolecular Screening has published recommended microplatespecifications for a variety of plate formats (seehttp://www.sbsonline.org).

A variety of plate readers are available for conducting assaymeasurements in multi-well plates including readers that measure changesin optical absorbance, emission of luminescence (e.g., fluorescence,phosphorescence, chemiluminescence, and electrochetniluminescence),emission of radiation, changes in light scattering, and changes in amagnetic field. U.S. Patent Application Publication 2004/0022677 andU.S. Pat. No. 7,842,246, respectively, of Wohlstadter et al. describesolutions that are useful for carrying out singleplex and multiplex ECLassays in a multi-well plate format. They include plates that comprise aplate top with through-holes that form the walls of the wells and aplate bottom that is sealed against the plate top to form the bottom ofthe wells. The plate bottom has patterned conductive layers that providethe wells with electrode surfaces that act as both solid phase supportsfor binding reactions as well as electrodes for inducingelectrochemiluminescence (ECL). The conductive layers may also includeelectrical contacts for applying electrical energy to the electrodesurfaces.

Despite such known methods and systems for conducting assays, improvedapparatuses, systems, methods, reagents, and kits for conductingautomated sampling, sample preparation, and/or sample analysis in amulti-well plate assay format are needed.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a method for focusing an opticalsensor to a spaced apart platform comprising the steps of: (a) providingat least a higher, middle and lower patterned surface, wherein themiddle patterned surface and the platform are aligned to each other andwherein a first distance between the higher and middle patternedsurfaces and a second distance between the middle surface and lowerpatterned surface are substantially equal; (b) obtaining a firstdifference in contrast values between the higher and middle patternedsurfaces with the optical sensor; (c) obtaining a second difference incontrast values between the middle and lower patterned surfaces with theoptical sensor; and (d) comparing the first and second differences incontrast values.

The invention further provides a focusing mechanism for an opticalsensor comprising at least a higher, middle and lower patterned surfacespaced apart from the optical sensor; wherein the middle patternedsurface is aligned to a target surface to be focused by the opticalsensor and the middle patterned surface, wherein a first distancebetween the higher and middle patterned surfaces and a second distancebetween the middle surface and lower patterned surface are substantiallyequal, wherein the optical sensor and the patterned surfaces are movedrelative to each other until a difference between a first and a seconddifferences in contrast values between the higher and middle patternedsurfaces and between the middle and lower patterned surfaces is lessthan a predetermined value; and wherein an illuminating source ispositioned to project light through the higher, middle and loweredpatterned surfaces toward the optical sensor.

The invention contemplates an instrument comprising: (a) a contactplatform, wherein the contact platform comprises a plurality ofinterrogation zones and each interrogation zone comprises at least apair of electrical contacts to apply a voltage potential to theinterrogation zone, (b) a controller operatively connected to a voltagesource, wherein the voltage source is connectable to one or more pairsof electrical contacts, and (c) a multiplexer connected to thecontroller and to the voltage source for selectively connecting thevoltage source to the pair of electrical contacts of a singleinterrogation zone or connecting the voltage source to the pairs ofelectrical contacts of more than one interrogation zones.

The instrument of the invention also includes: (a) a contact platform,wherein the platform comprises a plurality of interrogation zones andeach interrogation zone comprises at least a pair of electrical contactsto conduct a voltage potential to the interrogation zone, (b) acontroller operatively connected to a voltage source, wherein thevoltage source is connectable to one or more pairs of electricalcontacts, and (c) a means connected to the controller and the voltagesource for switching from a first connection between the voltage sourceand the electrical contacts of a single interrogation zone to a secondconnection between the voltage source and the electrical contacts of oneor more interrogation zones.

The instrument is preferably adapted to interrogate samples contained ina multi-well plate, and comprises: (a) a carriage frame configured tosupport the multi-well plate and the carriage frame is movable relativeto a contact platform, wherein the multi-well plate comprises aplurality of wells, wherein the wells are arranged in a M×N matrix, andwherein the contact platform comprises a plurality of interrogationzones, wherein each interrogation zone comprises at least a pair ofelectrical contacts to conduct a voltage potential to at least one well;(b) a controller operatively connected to a motor to move the carriageframe relative to the contact platform and operatively connected to avoltage source, wherein the voltage source is connectable to one or morepairs of electrical contacts; and (c) a multiplexer connected to thecontroller and to the voltage source for selectively connecting thevoltage source to the pair of electrical contacts of a singleinterrogation zone or connecting the voltage source to at least one pairof electrical contacts of more than one interrogation zones.

Another embodiment of the invention is a method for interrogatingsamples contained in a multi-well plate having a M×N matrix of wellscomprising the steps of (a) providing a contact platform having aplurality of interrogation zones, (b) providing at least a pair ofelectrical contacts for each interrogation zone, wherein eachinterrogation zone is adapted to interrogate a single well, (c)selectively applying a voltage potential to: (i) one interrogation zoneto interrogate one or more wells simultaneously or (ii) a plurality ofinterrogation zones to interrogate a plurality of wells, and (d) movingthe multi-well plate relative to the platform to interrogate additionalwells.

In a specific embodiment, the invention includes an instrument forconducting luminescence assays in a multi-well plate. The instrumentcomprises a light detection subsystem and a plate handling subsystem,wherein the plate handling subsystem comprises:

-   -   (a) a light-tight enclosure comprising a housing and a removable        drawer, wherein        -   (x) the housing comprises a housing top, a housing front,            one or more plate introduction apertures, a detection            aperture, a sliding light-tight door for sealing the plate            introduction apertures, and a plurality of alignment            features, wherein the housing is adapted to receive the            removable drawer, and        -   (y) the removable drawer comprises:            -   (i) an x-y subframe including plurality of companion                alignment features configured to mate and engage with                the plurality of alignment features to align the                removable drawer within the housing relative to the                light detection subsystem, wherein a weight of the                removable drawer is supported by the housing top;            -   (ii) one or more plate elevators with a plate lifting                platform that can be raised and lowered, wherein the one                or more plate elevators are positioned below the plate                introduction apertures;            -   (iii) a plate translation stage for translating a plate                in one or more horizontal directions, wherein the stage                comprises a plate carriage for supporting the plate, the                plate carriage has an opening to allow the plate                elevators positioned below the plate carriage to access                and lift the plate, and the plate translation stage is                configured to position plates below the detection                aperture and to position the plates above the plate                elevators; and    -   (b) one or more plate stackers mounted on the housing top, above        the plate introduction apertures, wherein the plate stackers are        configured to receive or deliver plates to the plate elevators;        and        -   wherein the light detection subsystem comprises a light            detector mounted on the enclosure top and coupled to the            detection aperture with a light-tight seal.

The instrument can be used to conduct luminescence assays in amulti-well plate, and comprises a plate handling subsystem including aplate carriage for supporting the multi-well plate, wherein the platecarriage comprises a frame and a plate latching mechanism. The platelatching mechanism comprises:

-   -   (a) a plate carriage ledge;    -   (b) a plate clamp arm perpendicular to the ledge and comprising        a proximate and a distal end relative to the ledge, wherein the        arm is attached to the frame at the proximate end and the arm is        rotatable in an x-y plane at the distal end, and the arm further        comprises an upper clamp including an angled surface configured        to engage with the plate;    -   (c) a plate positioning element comprising a rod, a pedal and a        spring, wherein the rod is substantially perpendicular to the        arm, substantially parallel to the ledge, and attached to the        distal end of the arm via the spring, and the pedal is attached        to the rod at an angle; and    -   (d) a plate wall substantially parallel to the arm and        substantially perpendicular to and disposed between the        positioning element and the ledge, the wall comprising (i) a        lower plate clamp configured to engage with a multi-well plate        skirt, and (ii) a lower plate clamp ramp configured to drive the        lower plate clamp toward the skirt.

The present invention is further directed to a method of engaging amulti-well plate in the instrument immediately discussed above. Themethod comprises the following steps:

-   -   (a) placing the plate on the frame;    -   (b) compressing the spring of the plate positioning element,        thereby pushing the pedal against the plate toward the ledge and        rotating the arm in the x-y plane toward the plate;    -   (c) contacting the upper clamp with the plate, thereby pushing        the plate toward the carriage wall;    -   (d) contacting the lower plate clamp with the skirt, thereby        locking the plate within the carriage.

Moreover, the invention provides an instrument for conductingluminescence assays in a multi-well plate, and comprises a platehandling subsystem including a plate carriage for supporting themulti-well plate, and a plate latching mechanism,

wherein the multi-well plate has at least a first, second, third andfourth side and wherein the first and third sides are substantiallyparallel to each other and the second and fourth sides are substantiallyparallel to each other,

wherein the plate carriage defines an aperture having a shapesubstantially the same as the multi-well plate and having dimensionssmaller than the multi-well plate to support a ledge positioned around aperimeter of the multi-well plate, wherein the plate carriage furthercomprises a first (501) and second (513) stop corresponding to the firstand second sides of the multi-well plate, respectively,

wherein the plate latching mechanism is movable from an openconfiguration to accept one multi-well plate to a clamping configurationto latch the multi-well plate to the plate carriage,

wherein the plate latching mechanism comprises a first latching member(509) biased to the clamping position and having a pedal (511) adaptedto push the first side of the multi-well plate toward the first stop anda plate clamp arm (502) biased to the clamping position and having abracket (503) pivotally connected to the plate clamp arm (502) and isadapted to push the second side toward the second stop (513), whereinthe first latching mechanism (509) is connected to the plate clamp arm(502), and

wherein the plate latching mechanism comprises at least one biased clamp(515) positioned proximate to second stop (513) to clamp to the skirt ofthe multi-well tray to the plate carriage.

Still further, the invention provides a system comprising

-   -   (i) a multi-well assay plate selected from the group consisting        of a single-well addressable plate or a multi-well addressable        plate; and    -   (ii) an apparatus configured to measure electrochemiluminescence        (ECL) from a single well of the single-well addressable plate        and from a grouping of wells of the multi-well addressable        plate.

The present invention further includes an apparatus for measuringluminescence from a multi-well plate of a plate type selected from thegroup consisting of a single well addressable plate or a multi-welladdressable plate, the apparatus comprising:

-   -   (i) a plate type identification interface for identifying the        plate type;    -   (ii) a plate translation stage for holding and translating the        multi-well plate in the x-y plane;    -   (iii) a plate contact mechanism comprising a plurality of        contact probes and positioned below the plate translation stage        and within the range of motion of the stage, wherein the        mechanism is mounted on a contact mechanism elevator that can        raise and lower the mechanism to bring the probes into and out        of contact with a bottom contact surface the plate when        positioned on the translation stage;    -   (iv) a voltage source for applying potential through the contact        probes to the plate; and    -   (v) an imaging system positioned above the plate translation        stage and in vertical alignment with the plate contact        mechanism, wherein    -   (a) the imaging system is configured to image a P×Q matrix of        wells, the plate contact mechanism is configured to contact the        bottom contact surface associated with the matrix and the plate        translation stage is configured to translate a plate to position        the matrix in alignment with the imaging system and plate        contact mechanism;    -   (b) the apparatus is configured to sequentially apply a voltage        to each well in the matrix of a single well addressable plate        and image the matrix; and    -   (c) the apparatus is configured to simultaneously apply a        voltage to each well in the matrix of a multi-well addressable        plate and image the matrix.

Also provided is a method for measuring luminescence from a single-welladdressable plate or a multi-well addressable plate, wherein the methodcomprises:

-   -   (a) loading a plate on the plate translation stage;    -   (b) identifying the plate as being a single well or multi-well        addressable plate;    -   (c) moving the plate translation stage to align a first P×Q        matrix of wells with the plate contact mechanism and imaging        system;    -   (d) raising the plate contact mechanism so that the contact        probes on the contact mechanism contact the bottom contact        surface associated with the P×Q matrix of wells;    -   (e) generating and imaging luminescence in the P×Q matrix by        sequentially applying voltage to each well in the group while        the group is imaged, if the plate is a single well addressable        plate;    -   (f) generating and imaging luminescence in the P×Q matrix by        simultaneously applying voltage to each well in the matrix while        the matrix is imaged, if the plate is a multi-well addressable        plate; and    -   (g) repeating steps (c) through (f) for additional P×Q matrices        in the plate.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1(a)-(b) show a front and rear view, respectively, of apparatus100 with a stylized cover and FIGS. 1(c)-(d) show the correspondingfront and rear views, respectively, of the apparatus without the cover.

FIGS. 2(a)-(c) show detailed views of the plate handling subsystem andlight detection subsystem.

FIG. 3 shows a view of the removable drawer of the plate handlingsubsystem within apparatus 100.

FIGS. 4(a)-(f) show various detailed views of the removable drawer 240and the subcomponents positioned within the drawer.

FIGS. 5(a)-(o) show detailed views of the plate carriage and platelatching mechanism.

FIGS. 6(a)-(b) show two alternative embodiments of an optical focusingmechanism that can be incorporated into the apparatus.

FIGS. 7(a)-(l) show detailed views of the plate contact mechanism.

FIGS. 8(a)-(c) show various components of the light detection subsystem.

FIG. 9 shows one non-limiting embodiment of a lens configuration thatcan be used in the light detection subsystem.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The Detailed Description section provides descriptions of certainembodiments of the invention that should not be considered limiting butare intended to illustrate certain inventive aspects. Unless otherwisedefined herein, scientific and technical terms used in connection withthe present invention shall have the meanings that are commonlyunderstood by those of ordinary skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. The articles “a” and “an”are used herein to refer to one or to more than one (i.e., to at leastone) of the grammatical object of the article. By way of example, “anelement” means one element or more than one element. Furthermore, aclaim which recites “comprising” allows the inclusion of other elementsto be within the scope of the claim; the invention is also described bysuch claims reciting the transitional phrases “consisting essentiallyof” (i.e., allowing the inclusion of other elements to be within thescope of the claim if they do not materially affect operation of theinvention) or “consisting of” (i.e., allowing only the elements listedin the claim other than ancillary elements or inconsequential activitieswhich are ordinarily associated with the invention) instead of the“comprising” term. Any of these three transitions can be used to claimthe invention.

Described herein is an apparatus for conducting assays in a multi-wellplate format that have one or more of the following desirableattributes: (i) high sensitivity, (ii) large dynamic range, (iii) smallsize and weight, (iv) array-based multiplexing capability, (v) automatedoperation; and (vi) ability to handle multiple plates. We also describecomponents and subsystems used in such an apparatus and methods of usingthe apparatus and subsystems. The apparatus and methods may be used witha variety of assay detection techniques including, but not limited to,techniques measuring one or more detectable signals. Some of them aresuitable for electrochemiluminescence measurements and, in particular,embodiments that are suitable for use with multi-well plates withintegrated electrodes (and assay methods using these plates) such asthose described in U.S. Publication 2004/0022677 and U.S. Pat. No.7,842,246, respectively, of Wohlstadter et al., and U.S. applicationSer. No. 11/642,970 of Glezer et al.

In a preferred embodiment, an apparatus is provided for conductingluminescence assays in multi-well plates. One embodiment comprises alight detection subsystem and a plate handling subsystem, wherein theplate handling subsystem includes a light-tight enclosure that providesa light-free environment in which luminescence measurements can becarried out. The enclosure includes a housing and a removable drawerthat is placed within the housing. The housing also includes a housingtop having one or more plate introduction apertures through which platescan be lowered onto or removed from a plate translation stage (manuallyor mechanically) within the drawer. A sliding light-tight door in thehousing is used to seal the plate introduction apertures fromenvironmental light prior to carrying out luminescence measurements. Thehousing further includes a detection aperture that is coupled to a lightdetector mounted on the housing top and one or more plate stackersmounted on the housing top above the plate introduction apertures,wherein the plate stackers are configured to receive or deliver platesto plate elevators within the removable drawer. The removable drawerincludes a plate translation stage for translating a plate horizontallyin the drawer to zones within the apparatus where specific assayprocessing and/or detection steps are carried out. The removable draweralso includes one or more plate elevators with a plate lifting platformthat can be raised and lowered within the drawer, wherein the plateelevators are positioned below the one or more plate introductionapertures. The plate translation stage is configured to position platesbelow the detection aperture and to position plates above the plateelevators on the plate lifting platforms.

The apparatus also includes a light detector which is mounted to thedetection aperture on the housing top (e.g., via a light-tight connectoror baffle). In certain embodiments, the light detector is an imaginglight detector such as a CCD camera and may also include a lens. Thelight detector may be a conventional light detector such as aphotodiode, avalanche photodiode, photomultiplier tube, or the like.Suitable light detectors also include arrays of such light detectors.Light detectors that may be used also include imaging systems such asCCD and CMOS cameras. The light detectors may also include lens, lightguides, etc. for directing, focusing and/or imaging light on thedetectors. In certain specific embodiments, an imaging system is used toimage luminescence from arrays of binding domains in one or more wellsof an assay plate and the assay apparatus reports luminescence valuesfor luminescence emitted from individual elements of the arrays. Thelight detector is mounted on the housing top with a light-tight seal.Additional components of the apparatus include plate contacts for makingelectrical contact to the plates and providing electrical energy toelectrodes in wells positioned under the light detector (e.g., forinducing ECL).

Specific embodiments of the apparatus of the invention are illustratedin the Figures. FIGS. 1(a)-(b) show a front and rear view, respectively,of apparatus 100 with a stylized cover, and FIGS. 1(c)-(d) show thecorresponding front and rear views, respectively, of the apparatuswithout the cover. As shown, e.g., in FIG. 1(c), the apparatus includesa light detection subsystem 110 and a plate handling subsystem 120. Amore detailed view is provided in FIGS. 2(a)-(b). The plate handlingsubsystem 120 includes a light tight enclosure 130 comprising a housing231 having a housing top 232, bottom 233, front 234, and rear 235. Thehousing also includes a plurality of alignment features and the housingis adapted to receive a removable drawer 240 comprising a removabledrawer front and consisting of a unitary casting element. The walls ofthe removable drawer define a rigid x-y subframe, 415 in FIG. 4(d),including a plurality of companion alignment features. When the draweris properly placed within the housing, the alignment and companionalignment features mate and engage, thereby aligning the drawer and itscomponents with the components of the light detection subsystem. Whenthe alignment/companion alignment features are engaged, the weight ofthe removable drawer is supported by the housing top. The removabledrawer 240 in the apparatus 100 depicted in FIGS. 1(a)-(b) is best shownin FIG. 3, being in the partially opened or closed position. Removabledrawer 240 is also illustrated in FIG. 4(a) carrying various internalsubsystems described in detail below and in FIG. 4(b) being installedwithin housing 231, where housing rear 235 and a housing side areomitted for clarity. FIG. 4(c) shows housing 231 with an opening andalignment features 405, 406, and 407 positioned and dimensioned toreceive removable drawer 240.

In one embodiment, the plate handling subsystem further comprises aplate sensor configured to detect a plate in the subsystem. Suitableplate sensors include, but are not limited to a capacitive sensor,contact switch, ultrasonic sensor, weight sensor, or an optical sensor,or a combination thereof.

Referring to FIG. 2(a), the housing top 232 also includes one or moreplate introduction (and ejection) apertures, 236 and 237, respectively,through which plates are lowered onto or removed from the platetranslation stage (manually or mechanically). A sliding light-tight door(shown in FIG. 2(c) as 239) is used to seal the plate introductionapertures 236, 237 from environmental light prior to carrying outluminescence measurements. Moreover, the housing top also includes anidentifier controller to read and process data stored to an identifieron the plates. In one embodiment, the identifier controller is a barcode reader (238) mounted via a light-tight seal over an aperture in thehousing top, where the bar code reader is configured to read bar codeson plates placed on the plate translation stage within the housing. In apreferred embodiment, the bar code on a plate is read once the plate hasbeen lowered into the drawer. In an alternative or additionalembodiment, the plates comprise an EEPROM or an RFID and the housing topand/or drawer includes an identifier controller suitable forcommunicating with each of these identifiers. In a further additionalembodiment, an identifier controller can be provided separately from theapparatus. In this embodiment, information stored to an identifierattached to a plate or associated with a plate or a set of plates istransferred to the apparatus via a computer and/or network attachedthereto and/or manually input via a user interface of the computerand/or network. In this regard, reference is made to U.S. applicationSer. Nos. 12/844,345 and 13/191,000, the disclosures of which areincorporated herein by reference.

The plate handling subsystem further includes one or more plate stackersmounted on the housing top 232 above the plate introduction apertures236, 237, wherein the plate stackers are configured to receive ordeliver plates to the plate elevators. The plate handling subsystemoptionally includes a heating and/or cooling mechanism (e.g., aresistance heater, a fan, heat sinks, or a thermoelectric heater/cooler)to maintain temperature of the subsystem under desired conditions. Itmay also include a humidity control mechanism (e.g., a humidifier and/ordehumidifier, or a desiccant chamber to maintain the humidity of thesubsystem under desired conditions.

A detailed view of the removable drawer of the plate handling subsystemis shown in FIG. 4. Referring to FIG. 4(a), the drawer includes (i) aplate elevator mechanism 400 with plate lifting platforms, 401 and 402,that can be raised and lowered; and (ii) a plate translation stage 403for translating a plate in one or more horizontal directions, whereinthe stage includes a plate carriage 404 for supporting the plate. Theplate carriage 404 preferably has an opening 420 to allow the plateelevators 400 positioned below the plate carriage 404 to access and lifta plate, and the plate translation stage 403 is configured to positionplates below the detection aperture on housing top 232 and below thelight detectors within the light detection system 110, and to positionthe plates above the plate elevators 400. The plate lifting platforms401, 402 of the plate elevator 400 preferably comprises a non-skidsurface to prevent shifting of the plate on the plate lifting platformduring movement in the apparatus. The plate translation stage 403 hashorizontal motions, e.g., motions on a substantially horizontal plane orin an X-direction and Y-direction for translating a plate horizontallyin the drawer to one or more regions within the apparatus where specificassay processing and/or detection steps are carried out. In onenon-limiting example, as illustrated in FIG. 4(e), plate translationstage 403 is movable in one horizontal direction along rail 422, andplate carriage 404 is movable on rail 424 on plate translation stage 403in an orthogonal horizontal direction. In a preferred embodiment, theplate translation stage has two axes of motion, x and y, and motorscoupled to the axes of motion allow for automated movement of plates onthe stage.

The inclusion of a removable drawer 240 in the light-tight enclosure 130enhances the serviceability and manufacturability of the apparatus. Inorder to ensure proper alignment of the drawer 240 within the housing231 and therefore, proper alignment of the subsystems within the drawer240 with the light detection subsystem 110, the housing includes aplurality of alignment features and the x-y subframe of the drawerincludes a plurality of companion alignment features configured to mateand engage with the alignment features of the housing. A cut-away viewof the drawer 240 placed within the housing 231 with housing rear 235and a housing side omitted for clarity and properly aligned with thelight detection subsystem 110 is shown in FIG. 4(b).

In a preferred embodiment, the alignment features of drawer 240 comprisea plurality of holes and the corresponding alignment features on housing231 comprise a plurality of pins sized to fit within the holes. As shownin FIG. 4(c), the housing 231 preferably includes at least threealignment pins, pins 405 and 406 being positioned on the housing front234, and pin 407, which is positioned on the opposite end of thehousing. Additional alignment features can be included in the housingand drawer, as necessary. Preferably, the alignment features arepositioned or calibrated relative to the housing top, such that theweight of the drawer 240 is supported by the housing top 232. Thecompanion alignment features on the drawer that are configured to mateand engage with alignment pins 405, 406, and 407, are shown in FIG. 4(d)as holes 408, 409, 410 (in the embodiment shown in FIG. 4(d), alignmentpin 405 mates and engages with hole 408, pin 406 mates and engages withhole 409, and pin 407 mates and engages with hole 410). In addition, thedrawer also includes alignment latches, 416 and 417 (shown in FIG. 4(a))that mate and engage with companion alignment catches, 418 and 419 (FIG.4(c)), to lock/unlock the drawer within the housing.

Due to the alignment features 405-407 and 408-410 being positioned orcalibrated to housing top 232, while removable drawer 240 is insertedinto housing 231 guided by X-Y frame 415, after removable drawer 240 isfully inserted into housing 231, the weight of drawer 240 and componentsthereon are supported by housing top 232. An advantage of this featureis that since light detection system 110 is also mounted on housing top232 any calibration or alignment of the subsystems on drawer 240 tolight detection system 110 can be carried out directly relative to thelight detection system 110, without having to taking into account anygap or spacing between drawer 240 and housing top 232.

One or more additional engagement/locking features can be included inthe housing and/or drawer, for example, as shown in FIG. 4(e), in whichspring loaded pin 411 is mounted to the drawer 240 and configured tomate and engage with a hole 412 positioned in the plate carriage 403. Inone embodiment, a solenoid is used to actuate a spring loaded pin, suchas pin 411. In the embodiment shown in FIG. 4(f), when the platecarriage and plate translation stage are aligned, the alignment featurein the plate translation stage, pin 411, mates and engages with acorresponding locking feature in the plate carriage, element 412, asshown in FIG. 4(f). These alignment and/or engagement features lock theplate carriage in place to protect the subassembly from damage, e.g.,during shipping and/or installation.

In a further preferred embodiment, as shown in FIGS. 4(c)-(d), thehousing top comprises an electrical connection contact mechanism 413,and the drawer front comprises a companion electrical connection,element 414, wherein the electrical connection and its companion areconfigured to mate and engage with one another upon proper insertion andalignment of the drawer within the housing.

Referring to FIG. 4(a), in a preferred embodiment, the plate carriagecomprises a carriage platform 404 and a plate latching mechanismconfigured to receive and engage an exemplary plate hereinafter labeledas 426 placed on the carriage platform 404, as shown in FIG. 5(a)-(b)(FIG. 5(a) shows a view of the plate carriage with a plate 426 locked inplace and FIG. 5(b) shows the same view with the components of the platelatching mechanism visible and engaged with the plate in a lockedposition). As shown in FIG. 5(b), the outside edges of the plate followa standard design convention for multi-well plates and include a skirt522 that surrounds and is at a height lower than the walls of the plate(an enlarged view is shown in FIG. 5(o)). In other words, skirt 522 ispositioned proximate the bottom of plate 426. The plate latchingmechanism is designed to push the outside edge of the skirt on twoorthogonal sides of the plate against two corresponding physical stopsin the plate carriage, to provide a defined and reproducible positioningof the plate in the carriage. The plate latching mechanism is alsodesigned to apply a downward physical force in defined locations on thetop of the plate skirt to reproducibly and fixedly hold the plate in thevertical dimension.

A view of the plate carriage 404 and plate latching mechanism with aplate 420 is shown in FIG. 5(a)-(b). A sequence illustrating theoperations of the plate latching mechanism is shown in FIGS. 5(c)-5(f)and discussed below. In a specific embodiment, the plate carriage 404supports a multi-well plate 426 (or a consumable having the samefootprint and external physical geometry as a multi-well/microtitreplate configured for use in an apparatus as described herein) having atleast a first, second, third and fourth side and wherein the first andthird sides are substantially parallel to each other and the second andfourth sides are substantially parallel to each other. The platecarriage 404 defines an aperture 420 having a shape substantially thesame as the multi-well plate 426 and having dimensions smaller than themulti-well plate to support a skirt or ledge 522 positioned around aperimeter of the multi-well plate 426. The plate carriage furthercomprises a first (501) and second (513) stop surface that when theplate 426 is fully latched, define the horizontal positions of the skirt522 on first and second sides of the multi-well plate, respectively. Theplate latching mechanism is movable from an open configuration, as bestshown in FIGS. 5(i) and 5(j) to accept a plate 426 to a clampingconfiguration to latch the plate to the plate carriage, as best shown inFIGS. 5(a) and 5(b).

The plate latching mechanism comprises (i) a first latching member (509)biased to the clamping position and consisting of a pedal 511, anactuating rod 510, and a spring 512, which provides the biasing forceand preferably has a high spring force. The pedal (511) is adapted topush the first side of the multi-well plate 426 toward the first stop501 and a plate clamp arm (502) also biased to the clamping position byspring 512, wherein the first latching mechanism (509) is connected tothe plate clamp arm (502). The plate latching mechanism further includes(ii) a bracket (503) pivotally connected to the plate clamp arm (502)and adapted to push the second side of plate 426 toward the second stop(513). The plate latching mechanism also comprises (iii) at least onebiased clamp (515) positioned proximate to second stop (513) to clamp tothe skirt 522 of the multi-well plate 426 to the plate carriage 404,thereby preventing vertical motion. Clamp 515 engages with the plateskirt and applies a downward force on the skirt of the plate. Thebracket (503) preferably comprises at least two legs (504, 506) and bothare in contact with the fourth side of the multi-well plate. At leastone leg (504, 506) comprises a ramp (507, 508) to apply both sidewaysforce towards the second stop and downward force on the skirt of themulti-well plate (as shown in FIGS. 5(e)-(i)).

The first latching member 509 comprises an actuating rod (510), which isbiased to the clamping position by a spring (512) and in the clampingposition extends past one edge of the plate carriage (as shown in FIG.5(c). During loading and unloading of plates, as the plate carriage 404is moved into alignment with a plate elevator, the extended portion 510a of actuating rod (510) is pushed against a physical stop in thehousing, e.g., the rear wall of drawer 240 or housing rear 235, whichpushes extended portion 510 a of rod (510) into the carriage, as bestshown in FIG. 5(d) where rod 510 is not yet engaged and FIG. 5(e) whererod 510 is pushed. It is noted that when plate carriage 404 is movedagainst the physical stop, rod 510 and both biased clamps 515 arepushed. FIGS. 5(d) and 5(i) only show the retraction of rod 510 forclarity. The movement of rod (510) forces the pedal 511 to retracttoward rod 510 to make room for plate 426. As shown in FIG. 5(c), pedal511 is a cantilever type arm that is attached to rod 510 and has theability to flex like a spring. A fulcrum 524 fixedly attached to platecarriage 404 forces pedal 511 to retract or move in the direction of thearrow shown in FIG. 5(d) as rod 510 is pushed inward. Fulcrum 526 canalso be located on the sheath 526 that covers first latch member 509, asbest shown in FIG. 5(a). Plate clamp arm 502 is connected preferablypivotally at one end 528 to rod 510, and connected preferably pivotallyat the opposite end 530 to plate carriage 404. Bracket 503 is pivotallyconnected to plate clamp arm 502 at pivot point 531. As best shown inFIG. 5(d), as rod 510 is pushed inward pedal 511 and plate clamp arm 502with bracket 503 are retracted or moved away from opening 420.

An advantage of connecting bracket 503 pivotally to plate clamp arm 502is that bracket 503 can rotate, preferably slightly relative to plateclamp arm 502, so that both legs 504 and 506 of bracket 503 can makecontact with plate 426 during the latching process.

As discussed above, when plate carriage 404 is moved against thephysical stop, rod 510 and both biased clamps 515 are pushed. Asextended portions 515 a of biased clamp 515 are pushed inward, thisaction lifts the biased end 515 b upward against the force of spring532. As biased end 515 b is lifted into an open position, it is sizedand dimensioned to accept skirt 522 of plate 426, and as biased clamp515 is released spring 532 forces biased end 515 b downward and clamponto skirt 522 to hold tray 426 against upward motions.

The apparatus further comprises an ejector (516) to release plate 426from the latching mechanism. Ejector 516 has an extended actuatingelement (521) and like actuating rod (510) also is pushed against a stopin the instrument as the plate carriage is placed in alignment with theplate elevators, such that the ejector moves the multi-well plate 426away from the second stop 513. The ejector 516 is preferablyspring-loaded by springs 514 and it optionally includes an over-travelpreventer 534. Ejector 516 when activated pushes tray 426 away from stop513, and when ejector 516 is activated rod 510 and biased clamps 515 arealso moved to the open position, so that tray 426 can be pushed awayfrom stop 513 and biased claim ends 515 b. Over-travel preventer 534 canelastically deform to absorb some of the motion of ejector Movement ofthe carriage plate 404 away from the plate loading/unloading position(i.e., in alignment with the plate elevators), reverses them movement ofrod (510) and ejector (516) and resets the latching mechanism into thelatched configuration.

Engagement of a multi-well plate 426 with the plate latching mechanismto lock the plate 426 in the plate carriage 404 is illustrated in FIGS.5(i)-(m). FIG. 5(i) is similar to FIG. 5(d) showing the first latchmember 509 with pedal 511 retracted and arm 502/bracket 503 in the openposition. The latching mechanism remains unengaged and in the openposition in FIG. 5(j), allowing a multi-well plate 426 to be placed overopening 420 within the plate carriage 404. In the open configurationdepicted in FIG. 5(j), pedal 511, clamp arm 502, bracket 503 and biasedclamp 515 are biased away from opening 420 to allow a plate 426 to beloaded into the plate carriage 404. As shown in FIG. 5(j), extendedportions 510 a and 515 a are all pushed inward by motion of platecarriage 404 against a back stop such as the back side of drawer 240 orhousing rear 235.

When a plate 426 is placed into the plate carriage 404 as shown in FIG.5(k) and plate carriage 404 moves away from the back stop, pedal 511moving away from fulcrum 524 and outward to push and bias tray 426against first stop 501. Plate clamp arm 502 also moves with rod 510,allowing bracket 503 to push tray 426 against second stop 513. As shownin FIG. 5(k), only leg 504 is contacting tray 426; however, due to thepivoting connection at pivot point 531, second leg 506 wouldautomatically and quickly contact tray 426 as bracket 503 rotates aboutpivot 531. Biased clamp 515, which is preferably spring loaded bysprings 532, engages the plate skirt 522 of the multi-well plate 426 onthe second side of the plate as shown in FIG. 5(l), bracket 503 alsoengages with and pushes down on the plate skirt 522. As discussed above,legs 504 and 506 of bracket 503 has ramp 507, 508 and angled as shown.As legs 504 and 506 pushes tray 426, ramp 507, 508 contact skirt 522 andpushes tray 426 in two directions: toward second stop 513 and downward.As shown in FIG. 5(m), biased clamp 515, engages with plate skirt 522.

In a preferred embodiment the plate carriage 404 also includes anoptical focusing mechanism used by an optical sensor in the apparatus,such as the light detectors within light detection system 110 describedabove to measure contrast and focus. The optical focusing mechanismincludes at least two, or preferably at least three, patterned surfacesat different heights relative to the plate carriage and, consequently,to a target surface for focusing (i.e., the bottom of the wells of a96-well plate 426 held in the plate carriage 404). The inventionincludes a method for imaging the plurality of surfaces and, based onthe image, calculating the magnitude and direction of the imageadjustment needed to bring the target surface into focus. In oneembodiment, contrast values are calculated for the image of each surfaceand the focus height is determined as the height at which the change incontrast with change in height is minimized or, alternatively, fallsbelow a predetermined threshold value.

In one embodiment, the plate carriage includes at least three patternedsurfaces each at differing heights relative to the plate carriage. Twoalternative embodiments of an optical focusing mechanism are shown inFIG. 6(a)-(b). In certain preferred embodiments the surfaces havepatterns of differential transparency (e.g., patterns etched or cut intoa non-transparent substrate or a patterned non-transparent ink or filmprinted on a transparent surface) so that the pattern can be imagedusing light transmitted through the substrate. In alternativeembodiments, the surfaces/patterns are not transparent and the patternsare imaged using a light source that reflects light off the surface.

The focusing mechanism includes at least a higher, middle and lowerpatterned surface spaced apart from the optical sensor, wherein themiddle patterned surface and the target surface are aligned tosubstantially the same planar level, wherein a first distance betweenthe higher and middle patterned surfaces and a second distance betweenthe middle surface and lower patterned surface are substantially equal,and wherein the optical sensor and the patterned surfaces are movedrelative to each other until a difference between a first pair ofcontrast values between the higher and middle pattern and a second pairof contrast values between the middle pattern and the lower pattern isless than a predetermined value of about ±2.0 dimensionless units, asexplained below. This difference may be ±3.0 or ±4.0, or as low as ±1.0.Higher value of contrast differences allow easier but less accuratefocusing, and lower value of contrast differences yields more difficultbut more accurate focusing.

As shown in FIGS. 6(a)-(b), the mechanism preferably includes aplurality of patterned surfaces, e.g., at least two and optionally threepatterned surfaces (601-603), and the patterned surfaces comprisesubstantially the same pattern, e.g., a grid pattern. The patternedsurfaces are preferably adjacent to one another in a grouping. In theembodiment shown in FIG. 6(a), the mechanism also includes anunpatterned surface 604. Preferably, each of the patterned surfaces arelocated on parallel planar planes. In a preferred embodiment, the middlepatterned surface is at a height effectively equivalent to a focusposition of a well in multi-well tray 426 filled with a predeterminedamount of fluid. The lower patterned surface is at a height that isabout 0.25 mm below the middle patterned surface and the upper patternedsurface is at a height that is about 0.25 mm above the middle patternedsurface. In one embodiment, the lower patterned surface is at a heightof about 4-4.75 mm above the plate carriage (i.e., above the carriageplatform that the plate rests on). Preferably, the lower patternedsurface is at a height of about 4.5-4.7 mm above the plate carriage, andmost preferably, the lower patterned surface is at a height of about4.6-4.7 mm above the plate carriage. The middle patterned surface is ata height of about 4.5-5.0 mm above the plate carriage, preferably, about4.7-4.9 mm above the plate carriage, and most preferably, about 4.7-4.8mm above the plate carriage. And the higher patterned surface is at aheight of about 4.75-5.10 mm above the plate carriage, preferably about4.8-5.0 mm above the carriage platform, and most preferably about4.85-4.95 mm above the plate carriage. It is noted that any one of thesurfaces 601, 602 and 603 can be the middle patterned surface, thehigher pattern surface, or the lower pattern surface. In a preferredembodiment, the optical focusing mechanism is adjacent to the platecarriage.

Therefore, the invention provides a method for focusing an opticalsensor to a target surface comprising the steps of (a) providing atleast a higher, middle and lower patterned surface 601-603, wherein themiddle patterned surface and the target surface are at the same focalheight and wherein a first distance between the higher and middlepatterned surfaces and a second distance between the middle surface andlower patterned surface are substantially equal; (b) obtaining a firstcontrast value difference between the higher and middle patternedsurfaces with the optical sensor; (c) obtaining a second contrast valuedifference between the middle and lower patterned surfaces with theoptical sensor; and (d) comparing the first and second contrast valuedifferences and determining if the target surface is in focus and/ordetermining the magnitude and direction of focus adjustment needed toplace the target surface in focus.

During operation, the plate translation stage 403 translates the platecarriage 404 to position the optical focusing mechanism over the contactmechanism shown in FIGS. 7(a)-7(c)(1), which includes a light source,such as light outlets 725-728 shown in FIG. 7(c)(1). Light outlets725-728 can be connected to a single light emitting diode (LED) or eachlight outlet may have its own LED or other light sources. The lightsource is illuminated and a beam of light is shown on the underside ofthe optical focusing mechanism, more specifically under surfaces601-603. Preferably, light outlets 725-728 provides even lighting forsurfaces 601-603. An optical sensor or camera in the light detectionsubsystem 110 therefore, images the optical focusing mechanism,calculates the differences in contrast values described above, anddetermines if the target is in focus and/or determines the magnitude anddirection of the focus adjustment needed to place the target surface infocus. Based on the calculation, the focus of the optical sensor isadjusted accordingly, either manually or automatically, e.g., throughthe use of a motorized focus adjustment. Preferably, the method alsoincludes the steps of adjusting the distance between the optical sensorand the target surface and repeating the steps of obtaining the firstand second contrast values and comparing those contrast values until adifference between the first and second contrast values are less than apredetermined value. A suitable calculation to determine the contrastvalue is to take a region or interest (ROI) of an image that is coveredby the dot pattern of the focus target, e.g., surface 601, 602 or 603 ora portion thereof. The average and the standard deviation of all of thepixels within that ROI are measured. The average (AVG) and standarddeviation (StDEV) to calculate the contrast value (% CV) of that ROI aremeasured or ascertained.% CV=(StDEV/AVG)×100

Then the % CV for each ROI (high and low) are then subtracted to createthe difference value that is reported to the operator. % CV as shownabove is a unit-less or dimensionless value.

A preferred predetermined value of the difference in % CV contrastvalues is determined as ±2.0 experimentally by comparing ECL value as afunction of defocus from nominal. The magnitude of this difference maychange depending on the contrast function. A certain amount of defocuswas acceptable without affecting ECL. The preferred value of ±2 iswithin this range. A smaller value, e.g., ±1.5 or ±1.0 would be moreaccurate but also more difficult to achieve during the focus operation.A larger value, e.g., ±3.0 or ±4.0 would be less accurate but easier toachieve. One of ordinary skilled in the art may balance accuracy andoperational difficulty according to the teachings of the presentinvention. Differences in contrast values between ±1.0 and ±4.0 arewithin the scope of the present invention.

Other methodology of calculating or ascertaining contrast values, suchas those discussed in “Contrast in Complex Images” by Eli Peli,published in the Journal of the Optical Society of America, No. 10,October 1990, at pages 2032-2040, can be used. This reference isincorporated by reference herein in its entirety.

Additionally, plate carriage 404 contains a plurality of referenceelements. One reference element comprises an electrically conductivebottom surface 536 disposed on a bottom surface of plate carriage 404,as shown in FIG. 5(n), which is used, during setup of the apparatus, totrain the positioning of the contact mechanism used to contact thebottom of plates 426 held in the plate carriage 404. The contactmechanism, described in more detail hereinbelow, includes a series ofspring loaded contact members and can be raised to contact a plate 426'sbottom surface, e.g., to initiate an ECL measurement. As shown in FIG.5(n), the conductive bottom surface 536 is on the underside of the platecarriage 404 and it is configured to be at the same height as a platebottom when a plate 426 is latched in the plate carriage 404. Duringapparatus setup or adjustment, the contact mechanism is raised until itreaches a height where the contact members touch surface 536, asdetected by electrically measuring the drop in resistance betweencontact members, signaling that the contact members have properlytouched conductive surface 536 and would properly contact the platebottoms during ECL measurements. This measured height is used to set thecontact mechanism height for contacting plates 426 held in the platecarriage 404.

Still further, the plate carriage 404 comprises another referenceelement (depicted in FIG. 5(c) as semicircular apertures cut into platecarriage 404, i.e., elements 517-520). A light source, such as lightoutlet or LED 722 in the contact mechanism is projected through eachaperture 517-522. Plate translation stage 403 moving in the horizontalplane discussed above position each aperture 517-522 above light outlet722 shown in FIG. 7(c)(1). The light projected through each aperture isimaged by the light detector in light detection system 110 to referencethe location of the plate carriage 404 in the x-y space of thehorizontal plane relative to other components of the apparatus. In apreferred embodiment, the reference elements comprise one or moreindentations or cut-outs, e.g., on the edge of the plate platform, e.g.,as shown in FIG. 5(c), at the two ends of reference surfaces/stops (501)and (503). Advantageously, the elements may also be imaged to confirm ifthe plate is in the correct orientation.

Light outlet 722 and light outlets 725-728 are preferably illuminated bya single LED. A suitable LED can be connected to light pipes orwaveguides to the light outlets. A suitable LED can have differentintensity outputs depending on the voltage applied. In one example, asillustrated in FIG. 7(h), LED 739 is connected to multiplexer 738.Microprocessor 729 can instruct multiplexer 738 to apply a first voltageto LED 739 to activate light outlet 722 and to apply a second voltage toLED 739 to activate light outlets 725-728. Alternatively, multiple LEDscan be used for the light outlets.

The plate handling subassembly also includes one or more shipping locksto lock the plate carriage in place during shipping, discussed above andbest illustrated in FIG. 4(e). In a preferred embodiment, the shippinglocks include solenoid driven pin 411 on removable drawer 240 beingreceived in hole 412 on plate translation stage 403. The plate carriage404 rides on rails 422, 424, and preferably comprises a clamp to lockthe carriage in place. Still further, the plate carriage 404 includes aplate orientation sensor, such as an accelerometer or electronicleveler, to ensure that a multi-well plate 426 placed on the platecarriage 404 is in the correct orientation.

The plate handling subassembly 120 also includes a plate contactmechanism that includes electrical contact probes mounted onto a platecontact elevator for raising the probes to contact electrical contactson the bottom of a multi-well plate 426 discussed above, that are inturn connected to electrodes in the wells of the plate. The contactprobes are used to apply the electrical potentials to electrodes in oneor more wells of a multi-well plate 426. The plate contact mechanism andthe imaging apparatus are in alignment, such that the electrical contactis made with the well or set of wells that is/are directly under, and inthe imaging field of, the imaging apparatus. The contact mechanism isshown in FIG. 7(a)-(b) and includes a contact mechanism platform 701comprising four interrogation zones 702-705, wherein each zone includesa pair of electrical contact probes to conduct a voltage potential tothe interrogation zone. Preferably, interrogation zones 702-705 arearranged in quadrants or 2×2 matrix. However, interrogation zones can bearranged in a linear manner or in any P×Q matrix, wherein P and Q areintegers and can be different from each other. As discussed in moredetail below, multi-well plate 426 usable in inventive instrument 100can be arranged in a M×N matrix, where the M×N matrix is larger than theP×Q matrix. As discussed above, P×Q matrix can be 12×8, 24×16, 48×32wells or any number of wells.

The apparatus also includes a controller operatively connected to avoltage source, wherein the voltage source is connectable to one or morepairs of electrical contact probes, and a multiplexer connected to thecontroller and to the voltage source for selectively connecting thevoltage source to the pair of electrical contact probes of a singleinterrogation zone or connecting the voltage source to the pairs ofelectrical contact probes of more than one interrogation zone. A blockdiagram showing the components of the controller is shown in FIG. 7(h),including microprocessor 729, connected to power source 730 and digitalanalog converter 731, which is connected to low pass filters 732 and733, current monitor 734, another optional power source 737, and analogdigital converter 736, and a multiplexer 738. The controller is alsooperatively connected to an LED 739, which is a component of the contactmechanism, discussed above.

The multiplexer 737 controlled by processor 729 directs the applicationof potential as identified above based on the type of plate used in theinstrument. If the multi-well plate 426 is configured to be analyzed onewell at a time, referred to herein as a single-well addressable plate,wherein a well of a plate corresponds to a zone of the contact mechanismplatform, the multiplexer 737 will direct the selective application ofpotential by electrically isolating each zone and selectively applying apotential only within a first zone. If, on the other hand, themulti-well plate is configured to be analyzed two or more wells at atime, referred to herein as a multi-well addressable plate, themultiplexer 737 will direct the selective application of potential byelectrically connecting two or more zones and selectively applying apotential within those two or more zones. In one embodiment, the platescomprise a bar code that includes plate configuration information andthe apparatus 100 comprises a bar code reader 238 that reads the plateconfiguration information and identities the type of plates positionedin the stacker.

In a preferred embodiment, the apparatus includes a plurality ofinterrogation zones 702-705 that are arranged in a P×Q matrix.Preferably, the P×Q matrix is a 2×2 matrix. The pairs of electricalcontact probes on the plate contact mechanism platform 701 preferablycomprise upstanding pins, e.g., spring-loaded pin. Still further, theapparatus preferably further includes an optical sensor, such as thelight detectors in the light detection system 110, positioned above theplatform 701 and the platform 701 includes a first alignment mechanismcomprising a light source, such as light outlet 722 projecting from theplatform toward the optical sensor to align the platform 701 relative tothe optical sensor. In one embodiment, the light source (e.g., an LED orother type of light bulb) is positioned under and shines light throughan aperture in the contact mechanism, e.g., through aperture (722) whichis centered in platform (701) as shown in FIG. 7(c)(1). The apparatusalso preferably includes a second alignment mechanism comprising aplurality of apertures located on the plate carriage frame (e.g.,elements 517-520 shown in FIG. 5(c)) and the light source 722 from theplatform 701 can be illuminated through these apertures and detected bythe optical sensor to further align the plate carriage frame with theplatform 701. The plurality of apertures can be positioned on at leasttwo sides of the plate carriage frame (see description above). Moreover,the apparatus further preferably includes a third alignment mechanismcomprising an electrical conductive surface located on the platecarriage frame (e.g., surface 536 in FIG. 5(n)) such that when theelectrical contacts on the platform are brought in contact with theelectrical conductive surface electrical current flows among theelectrical contacts on the platform to indicate a predetermined distancebetween the electrical contacts and the plate carriage frame. Theapparatus preferably includes a fourth alignment/focusing mechanismcomprising patterned focusing targets (e.g., surfaces 601-603 in FIGS.6(a) and 6(b)) and the contact mechanism platform includes one or morelight sources for passing light through the patterns to enable imagingof the patterns, discussed above. The light source(s) may be a lightsource under aperture (722) as described above. Optionally, a pluralityof light sources (e.g., LEDs or other types of light bulbs) may be usedto generate a wider and more even light field, e.g., the four LEDs(725-728) embedded in the plate contact mechanism platform as shown inFIG. 7(c)(1).

In a preferred embodiment, the apparatus is adapted to interrogatesamples contained in a multi-well plate, wherein the multi-well platecomprises a plurality of wells arranged in an M×N matrix, and theapparatus includes a carriage frame configured to support the multi-wellplate, wherein the carriage frame is movable relative to a contactmechanism platform comprising a plurality of interrogation zones,wherein each interrogation zone comprises at least a pair of electricalcontact probes to apply a voltage potential to at least one well. Theapparatus also includes a controller operatively connected to a motor tomove the carriage frame relative to the platform and operativelyconnected to a voltage source, wherein the voltage source is connectableto one or more pairs of electrical contacts, and a multiplexer connectedto the controller and to the voltage source for selectively connectingthe voltage source to the pair of electrical contact probes of a singleinterrogation zone or connecting the voltage source to at least one pairof electrical contact probes of more than one interrogation zones.Preferably, the interrogation zones are arranged in a P×Q matrix and theM×N matrix is larger than the P×Q matrix, which can be a 2×2 matrix.Preferably, each interrogation zone is sized and dimensioned tointerrogate one well on multi-well plate 426.

Preferably, the electrical contact probes on the contact mechanismplatform include a plurality of working electrode contact probes thatare selectively connected by the controller to the voltage source todetermine the number of wells to interrogate. In one embodiment, aworking electrode probe is connected to the working electrode in onewell, or alternatively, one working electrode probe is connected to theworking electrode in a plurality of wells. The working terminalselectrode probes that are not connected can be electrically isolated inthe multiplexer when not in use, thereby allowing a plurality of workingelectrode probes (e.g., 4 probes) to be used to apply potential to aplurality or working electrodes in a plurality of wells, one well at atime (e.g., applying potential to a group of 4 wells, one well at atime). The electrical contacts on the platform can further comprise aplurality of counter electrode probes that are electrically connected toat least one electrical ground. In one embodiment, the bottom electricalcontacts of the multi-well plate that are connected to the counterelectrode probes on the platform for a plurality of wells areelectrically connected. Alternatively, the bottom electrical contacts ofthe multi-well plate that are connected to the counter electrode probeson the platform for all the wells are electrically connected. Stillfurther, the bottom electrical contacts of the multi-well plate that areconnected to the counter electrode probes on the platform for at leastone well can be electrically isolated. The controller can interrogateP×Q or fewer number of wells simultaneously.

Referring to FIGS. 7(c)(2)-(g), the contact mechanism platform 701includes a plurality of working contact probes 706-713 and countercontact probes, 714-721. As shown in FIG. 7(c)(2), if the controller 709is configured to electrically connect two or more interrogation zones,then the instrument 100 selectively applies a potential within two ormore zones, e.g., zones 703 and 704, thereby applying a potential acrossworking electrode contact probes 706 and 710 and 709 and 713,respectively and connecting counter electrode contact probes 714-717 and718-721. The connections of the counter electrodes at platform 701 andplate 426 are discussed below. Also as discussed below, only one workingcontact electrode and one counter contact electrode are necessary. Twoof each are connected to provide a redundancy for the system, so that anECL signal is generated even when one electrode fails.

Alternatively, if the switching mechanism is configured to electricallyisolate each zone then the instrument selectively applies a potentialwithin a first zone, e.g., as in FIG. 7(d), wherein zone 703 is isolatedand an electrical potential is applied across working electrode contactprobes 706 and 710. In one embodiment, all counter electrode contactprobes 714-717 and 718-721, which are connected to ground, areelectrically connected at platform 701. As discussed below in connectionwith FIG. 7(k), the counter electrode contact probes for each well areisolated by the counter electrodes on the bottom of plate 426. In theexample shown in FIG. 7(d), the well directly above zone 703 has acounter electrode that connects to counter electrode contact probes 718and 719, but isolates from the other counter electrode contact probes onplatform 701. Alternatively, the counter electrodes for eachinterrogation zone can be isolated at platform 701.

Similarly, FIGS. 7(e)-(g) illustrate how the contact mechanism isconfigured to apply a potential within a first zone, 702 (FIG. 7(e)),705 (FIG. 7(f)), and 704 (FIG. 7g ), and a potential is applied acrossworking contact probes 707 and 712 (in FIG. 7(e)), 708 and 711 (in FIG.7(f)), or 709 and 713 (in FIG. 7(g)), respectively, while countercontact probes 714-717 and 718-721 are electrically connected atplatform 701, but the counter contact probes for each interrogation zoneare isolated by the counter electrode on the well on plate 426 directlyabove each interrogation zone. Preferably, the contact probes are eachindependently spring-loaded contacts members, e.g., contact pins.

In a preferred embodiment, the multi-well plate 426 comprises bottomelectrical contacts on a bottom surface of the plate for each well,wherein the bottom electrical contacts are configured to contact thepair(s) of electrical contact probes on the platform 701. The bottomelectrical contacts include counter electrode contacts that areconnected to counter electrodes in the wells of the plate and workingelectrode contacts that are connected to working electrodes in the wellsof the plate. Each well includes at least one working and one counterelectrode, which depending on the plate format, may be electricallyconnected (bussed) or electrically independent of the working andcounter electrodes in other wells of the plate.

A non-limiting set of exemplary bottom electrical contact patterns areshown in FIGS. 7(i)-(l), wherein FIG. 7(i) shows the pin contactconfiguration of platform 701 substantially similar to FIG. 7(c)(2).FIG. 7(k) shows an overlap of the bottom electrical contacts underexemplary four wells that overlay interrogation zones 702-705. Each wellhas bottom counter electrode 740 having an exemplary “Z-shape” and twoworking electrodes 742 and 744. Bottom counter electrodes 740 are notelectrically connected to each other, and hence the counter electrodesfor each well or each interrogation zone are separated or isolated atplate 426.

For zone 703, Z-shape bottom counter electrode 740 connects to counterelectrodes 718 and 719. Bottom working electrodes 742 and 744 areconnected to working electrodes 710 and 706, respectively.

For zone 705, Z-shape bottom counter electrode 740 connects to counterelectrodes 720 and 721. Bottom working electrodes 742 and 744 areconnected to working electrodes 711 and 708, respectively. Zones 702 and704 are similarly connected.

The next electrical connection is to the inside of the well itself. Asillustrated in FIG. 7(l), each well in this example has well workingelectrode 750 and well counter electrodes 752 and 754. Here, wellworking electrode 750 has a Z-shape and connects to both bottom workingelectrode 742 and 744, and well counter electrodes 752 and 754 areconnected to bottom counter electrode 740.

For zone 705, working electrodes 711 and 708 on platform 701 areconnected to bottom electrodes 742 and 744 and well working electrode750 for each well. Counter electrodes 720 and 721 on platform 701 areconnected to bottom counter electrode 740 and well counter electrodes752 and 754 for each well. The Z-shapes for bottom electrode 740 andwell electrode 750 are designed to endure sufficient electrical contact.Any shape can be used and the present invention is not limited to anyparticular shape.

As shown in the above discussion, each well and each interrogation zonehas two working electrodes, e.g., 708 and 711 for zone 705, and twocounter electrodes, e.g., 720 and 721 for zone 705. Both workingelectrodes and both counter electrodes are electrically connected to awell as shown above only one pair of working and counter electrodes isnecessary to conduct ECL potential to a well. The other pair is forredundancy, in case one or more electrode malfunctions.

It is further noted that in the example discussed above in connectionwith FIGS. 7(i), 7(k) and 7(l) where each well can be interrogatedindividually, the working electrodes for each interrogation zone andwell are isolated at platform 701 and multiplexer 738, and the counterelectrodes for each interrogation zone and well are isolated at plate426 and its bottom electrodes and well electrodes.

FIG. 7(j) illustrates an example where four wells overlayinginterrogation zones 702-705 can be interrogated at the same time usingthe contact pins or electrodes from the same platform 701. As shown,this multi-well plate 426 has bottom working electrode 760 overlayingworking electrodes 707, 708 and 709. Tray 426 also has bottom counterelectrode 762 overlaying at least counter electrode 719, 720, 715 and716. Bottom working electrode 760 and bottom counter electrode 762 areelectrically connected upward to all four wells. Activating one or moreworking electrodes 707, 708 and 709 and one or more counter electrodes719, 720, 715 and 716 would provide an ECL potential to all four wells.Redundancy is also provides by the plurality of available working andcounter electrodes.

According to an embodiment of the present invention, the plate bottomcomprises internal electrical contacts conduits connected to the bottomelectrical contacts to conduct the voltage potential to within thewells. In one embodiment, the bottom electrical contacts for at leastone well are electrically isolated from the bottom electrical contactsfor adjacent wells and optionally, the internal electrical contactsconduits for at least one well can be electrically isolated from thebottom electrical contacts for adjacent wells. Reference is made to U.S.Pat. No. 7,842,246 and U.S. Application No. 20040022677 (both entitled“Assay Plates, Reader Systems and Methods for Luminescence TestMeasurements”, filed on Jun. 28, 2002, hereby incorporated byreference), which discloses additional embodiments of plate bottoms thatcan be interrogated by the contact mechanism disclosed herein.

Therefore, the invention provides a method for interrogating samplescontained in a multi-well plate having a M×N matrix of wells comprisingthe steps of (a) providing a plate contact mechanism platform having aplurality of interrogation zones, (b) providing at least a pair ofelectrical contact probes (e.g., a working electrode contact probe and acounter electrode contact probe) for each interrogation zone, whereineach interrogation zone is adapted to interrogate a single well, (c)selectively applying a voltage potential to: (i) one interrogation zoneto interrogate one or more wells simultaneously or (ii) a plurality ofinterrogation zones to interrogate a plurality of wells, and (d) movingthe multi-well plate relative to the platform to interrogate additionalwells. A single well can be interrogated or a M×N number of wells can beinterrogated (wherein M×N is larger than the P×Q matrix). The method canalso include the step of (e) controlling the application of voltagepotential in step (c) by selecting at least one positive active contactprobe (e.g., the working electrode probe) of the pairs of the electricalcontact probes on the platform to connect to the voltage potential. Step(e) can also include the step of electrically isolating at least onepositive active contact probe not connected to the voltage potential.The method can also include step (f), providing bottom electricalcontacts on a bottom surface of the multi-well plate and optionally, (g)electrically isolating at least one ground contact probe (e.g., thecounter electrode probe) from the bottom electrical contacts.Optionally, all ground contact probes from the bottom electricalcontacts are isolated from each other.

As described above, the apparatus can be used to measure luminescencefrom two alternative types of multi-well plates, a single-welladdressable plate (i.e., a plate that is interrogated by the apparatusone well at a time), and/or a multi-well addressable plate (i.e., aplate that is interrogated by the apparatus one sector at a time,wherein a sector is a grouping of adjacent wells). Various types ofmulti-well plates including single-well and multi-well addressableplates are described in U.S. Pat. No. 7,842,246 and U.S. Application No.20040022677 (both entitled “Assay Plates, Reader Systems and Methods forLuminescence Test Measurements”, filed on Jun. 28, 2002, herebyincorporated by reference). The plates of the invention include severalelements, including but not limited to, a plate top, a plate bottom, aplurality of wells, working electrodes, counter electrodes, referenceelectrodes, dielectric materials, electrical connections, conductivethrough holes, and assay reagents. The wells of the plate are defined byholes/openings in the plate top and the plate bottom can be affixed tothe plate top, directly or in combination with other components, and theplate bottom can serve as the bottom of the well. One or more assayreagents can be included in wells and/or assay domains of a plate. Thesereagents can be immobilized or placed on one or more of the surfaces ofa well, preferably on the surface of an electrode and most preferably onthe surface of a working electrode. The assay reagents can be containedor localized by features within a well, e.g., patterned dielectricmaterials can confine or localize fluids. The plate top preferablycomprises a unitary molded structure made from rigid thermoplasticmaterial such as polystyrene, polyethylene or polypropylene. The platebottom preferably includes electrodes (e.g., working and/or counterelectrodes) that comprise carbon, preferably carbon layers, morepreferably screen-printed layers of carbon inks. In another preferredembodiment, the plate bottom includes electrodes comprised of a screenprinted conducting ink deposited on a substrate.

A single well addressable plate includes a plate top having plate topopenings and a plate bottom mated to the plate top to define wells ofthe single well addressable plate, the plate bottom comprising asubstrate having a top surface with electrodes patterned thereon and abottom surface with electrical contacts patterned thereon, wherein theelectrodes and contacts are patterned to define a plurality of wellbottoms of the single well addressable plate, wherein a pattern within awell bottom comprises: (a) a working electrode on the top surface of thesubstrate, wherein the working electrode is electrically connected to anelectrical contact; and b) a counter electrode on the top surface of thesubstrate, wherein the counter electrode is electrically connected withthe electrical contact, but not with an additional counter electrode inan additional well of the single well addressable plate. Preferably, theelectrodes and contacts of a single-well addressable plate areindividually addressable.

A multi-well addressable plate includes a plate top having plate topopenings and a plate bottom mated to the plate top to define wells ofthe multi-well addressable plate, the plate bottom comprising asubstrate having a top surface with electrodes patterned thereon and abottom surface with electrical contacts patterned thereon, wherein theelectrodes and contacts are patterned to define two or moreindependently addressable sectors of two or more jointly addressableassay wells, each sector comprising two or more wells with: (a) jointlyaddressable working electrodes on the top surface of the substrate,wherein each of the working electrodes is electrically connected witheach other and connected to at least a first of the electrical contacts;and (b) jointly addressable counter electrodes on the top surface of thesubstrate, wherein each of the counter electrodes is electricallyconnected with each other, but not with the working electrodes, andconnected to at least a second of the electrical contacts. In oneembodiment, the independently addressable sectors include less than 50%of the wells of the multi-well addressable plate, more preferably lessthan 20% of the wells of the multi-well addressable plate. Theindependently addressable sectors can comprise a 4×4 array of wells or a2×3 array of independently addressable sectors. Alternatively, theindependently addressable sectors can comprise one or more rows or oneor more columns of wells.

A single-well or multi-well addressable plate can be a 4 well plate, 6well plate, 24 well plate, 96 well plate, 384 well plate, 1536 wellplate, 6144 well plate or 9600 well plate. The electrodes of eitherplate format comprise carbon particles and they can further comprise aprinted conductive material, wherein one or more of the electrodescomprise a plurality of assay domains formed thereon. The plurality ofassay domains can include at least four assay domains, preferably sevendomains, and more preferably at least ten assay domains, and theplurality of assay domains can be defined by openings in one or moredielectric layers supported on the working electrodes. Plates that canbe used in the apparatus are available from Meso Scale Discovery(Rockville, Md.; www.mesoscale.com) and include but are not limited tothe following multi-well addressable plates (Meso Scale Discoverycatalog numbers): L15XA-3, L15XB-3, L15AA-1, L15AB-1, L15SA-1, L15SB-1,L15GB-1, L45XA-3, L45-XB-3, N45153A-2, N45153B-2, N45154A-2, andN45154B-2; and the following single-well addressable plates (Meso ScaleDiscovery catalog numbers): L55AB-1, L55SA-1, L55XA-1, and L55XB-1.

Accordingly, the apparatus measures luminescence from a multi-well plateby first detecting the plate type in the apparatus, e.g., by reading thebar code on the multi-well plate which includes plate configurationinformation, aligning the contact mechanism and imaging apparatus suchthat the interrogation zone or zones are directly under and in theimaging field of the imaging apparatus, and directing the selectiveapplication of potential by (a) electrically isolating eachinterrogation zone of the contact mechanism and selectively applying apotential only within a first zone (for a single-well addressableplate); or (b) electrically connecting two or more zone and selectivelyapplying a potential within those two or more zone (for a multi-welladdressable plate). If a multi-well addressable plate is being used inthe apparatus, the imaging system and contact mechanism are aligned withan interrogation zone that corresponds to a grouping or sector ofadjacent wells, e.g., a grouping of four adjacent wells, and theapparatus selectively applies a voltage to all wells of that sector. Theapparatus then moves the plate via the plate translation stage toreposition the contact mechanism and imaging system with an additionalinterrogation zone that corresponds to an additional sector or groupingof wells, and selectively applies a voltage to the wells of thatadditional sector. If a single well addressable plate is being used inthe apparatus, the imaging system and contact mechanism are aligned withan interrogation zone that corresponds to a grouping or sector ofadjacent wells, e.g., a grouping of four adjacent wells, and theapparatus selectively applies a voltage to each well of that sector oneat a time. Likewise, the plate is moved via the plate translation stageto reposition the contact mechanism and imaging system with anadditional interrogation zone that corresponds to an additional sectorof wells to interrogate each well of that additional sector one at atime.

In a specific embodiment, the apparatus can measure luminescence from asingle well addressable plate or a multi-well addressable plate, whereinthe apparatus includes:

-   -   (i) a plate type identification interface for identifying the        plate type;    -   (ii) a plate translation stage for holding and translating the        multi-well plate in the x-y plane;    -   (iii) a plate contact mechanism comprising a plurality of        contact probes and positioned below the plate translation stage        and within the range of motion of the stage, wherein the        mechanism is mounted on a contact mechanism elevator that can        raise and lower the mechanism to bring the probes into and out        of contact with a bottom contact surface the plate when        positioned on the translation stage;    -   (iv) a voltage source for applying potential through the contact        probes to the plate; and    -   (v) an imaging system positioned above the plate translation        stage and in vertical alignment with the plate contact        mechanism, wherein    -   (a) the imaging system is configured to image a P×Q matrix of        wells, the plate contact mechanism is configured to contact the        bottom contact surface associated with the matrix and the plate        translation stage is configured to translate a plate to position        the matrix in alignment with the imaging system and plate        contact mechanism;    -   (b) the apparatus is configured to sequentially apply a voltage        to each well in the matrix of a single well addressable plate        and image the matrix; and    -   (c) the apparatus is configured to simultaneously apply a        voltage to each well in the matrix of a multi-well addressable        plate and image the matrix.

Preferably, the P×Q matrix is a 2×2 array of wells. The imaging systemcan collect a separate image for each sequential application of voltageto each well in the matrix of a single well addressable plate. The platetype identification interface can include a bar code reader, an EPROMreader, an EEPROM reader, or an REID reader, or alternatively, the platetype identification interface comprises a graphical user interfaceconfigured to enable a user to input plate type identificationinformation.

Therefore, a method for measuring luminescence from a single welladdressable plate or a multi-well addressable plate using such anapparatus comprises:

-   -   (a) loading a plate on the plate translation stage;    -   (b) identifying the plate as being a single well or multi-well        addressable plate;    -   (c) moving the plate translation stage to align a first P×Q        matrix of wells with the plate contact mechanism and imaging        system;    -   (d) raising the plate contact mechanism so that the contact        probes on the contact mechanism contact the bottom contact        surface associated with the P×Q matrix of wells;    -   (e) generating and imaging luminescence in the P×Q matrix by        sequentially applying voltage to each well in the group while        the group is imaged, if the plate is a single well addressable        plate;    -   (f) generating and imaging luminescence in the P×Q matrix by        simultaneously applying voltage to each well in the matrix while        the matrix is imaged, if the plate is a multi-well addressable        plate; and    -   (g) repeating steps (c) through (f) for additional P×Q matrices        in the plate.

The removable drawer may include a light source (e.g., an LED) locatedunderneath the detection aperture and below the elevation of platetranslation stage. In one embodiment, this light source or plurality oflight sources are components of the plate contact mechanism. Asdescribed above in reference to the optical focusing mechanism, thelight source(s) in the contact mechanism are used in connection with theoptical focusing mechanism to adjust the contrast and focus of the lightdetector relative to a plate.

In an additional embodiment, one or more light sources) can also be usedin connection with fiducial holes or windows to correct for errors inplate alignment. Light from the light source is passed through thefiducials and imaged on the imaging apparatus so as to determine thecorrect for the alignment of the plate. Advantageously, plates formedfrom plate bottoms mated to a plate top (e.g., plates with screenprinted plate bottoms mated to injection-molded plate tops as describedin copending U.S. Applications 2004/0022677 and 2005/0052646) includefiducials patterned (e.g., screen printed) or cut into the plate bottomto correct for misalignment of the plate bottom relative to the platetop. In one specific embodiment, the plate top on such a plate includesholes (e.g., in the outside frame of the plate top) aligned withfiducials on the plate bottom to allow imaging of the fiducials.Accordingly, the imaging of light generated under a plate may be used tocommunicate the exact position of the plate to the image processingsoftware and also to provide for a camera focus check. The plate maythen be realigned using a two-axis positioning apparatus. Thus, theapparatus may process plates via a plate positioning method comprising:(1) providing a plate having light-path openings; (2) illuminating theplate from the bottom; (3) detecting light coming through light-pathopenings; and (4) optionally, realigning the plate.

In a preferred embodiment, the contact mechanism platform includes afirst alignment feature 722 and the light detection subsystem comprisesa camera positioned above the platform which is adjustable relative tothe first alignment feature. Preferably, the first alignment feature islight source, e.g., an LED. The camera in the light detection subsystemis adjustable relative to the alignment feature in the x-y plane. Theplatform can further include a plurality of additional alignmentfeatures, e.g., at least one additional alignment feature in eachquadrant, and the camera position is adjustable relative to eachadditional alignment feature. The additional alignment features cancomprise a light source, e.g., an LED. Therefore, as described above,the apparatus may confirm proper alignment of the contact mechanism andthe detection aperture using the optical focusing mechanism by: (1)illuminating the contact mechanism alignment features; (2) detectinglight coming from the alignment features; and (4) optionally, realigningthe plate translation stage, the light detector, and/or the contactmechanism. In one preferred embodiment, the apparatus confirms properalignment of the contact mechanism before making contact with the plateand then the plate position is confirmed by detecting light coming fromlight-path openings in the plate and realigning the plate as needed.

As illustrated in FIG. 7(a)-(b), the height of the contact mechanismplatform is adjustable because the platform further includes a shaft 723driven by a gear mechanism 724, in one embodiment, the gear mechanismcomprises a worm gear. In a preferred embodiment, the platform comprisesa plate surface area sized to accommodate a microtitre plate, e.g.,multi-well plate, and the platform further includes a spillagecollection area surrounding the plate surface area to protect componentsof the drawer from accidental spills of fluid that may be containedwithin the multi-well plate.

The light detection subsystem of the apparatus comprises a lightdetector that can be mounted to a detection aperture on the housing topvia a light-tight connector or baffle. In certain embodiments, the lightdetector is an imaging light detector such as a CCD camera and it alsoincludes a lens. A light detection subsystem is shown in FIG. 8(a). Thesubsystem includes a light detector housing 801 surrounding the lightdetector (not shown) and attached to the housing top via a castcomponent 802 that is bolted to the housing top over the detectionaperture. Above the cast component sits a buckle or clamp 803 thatincludes a theta adjustment mechanism comprised of screw 804 and gear805, illustrated in FIG. 8(b). The camera focusing mechanism is alsoconfigured to focus the camera in the x, y, and z directions as needed,either manually, via motorized elements, or both. The light detectionsubsystem further includes one or more light-fighting elements toprevent light leakage within the light detection subsystem or at thejuncture between the light detection subsystem and the housing top. Forexample, molded rubber or other compressible materials can be sandwichedbetween joined components to prevent light leakage. In addition, thelight detector housing includes one or more vents and/or coolingelements to cool the light detector within the housing. In oneembodiment, the housing includes an intake vent and an exhaust vent,each positioned on the opposite ends of the housing. Additional ventscan be positioned in the housing. In a preferred embodiment, the intakevent is sized to match a cooling fan positioned within the housing.

A lens, coupled to a camera, is used to provide a focused image ofluminescence generated from plates in the light-tight enclosure. Adiaphragm sealed to the lens and a detection aperture in the top ofenclosure, and allows the imaging system to image light from enclosurewhile maintaining the enclosure in a light-tight environment protectedfrom environmental light. Suitable cameras for use in the imaging systeminclude, but are not limited to, conventional cameras such as filmcameras, CCD cameras, CMOS cameras, and the like. CCD cameras may becooled to lower electronic noise. Preferably, the lens is a highnumerical aperture lens which may be made from glass or injection-moldedplastic. The imaging system may be used to image one well or multiplewells of a plate at a time. The light collection efficiency for imaginglight from a single well is higher than for imaging a group of wells dueto the closer match in the size of the CCD chip and the area beingimaged. The reduced size of the imaged area and the increase incollection efficiency allows for the use of small inexpensive CCDcameras and lenses while maintaining high sensitivity in detection.

If high resolution is not required, the sensitivity of the measurementcan be improved by using hardware binning on the CCD during imagecollection, which effectively reduces the electronic read noise per unitarea. Preferred binning depends on the field of view, demagnification,and size of the CCD pixels. In a preferred embodiment, the lightdetector comprises a camera with a CCD having 512×512 pixels, with eachpixel size being 24×24 microns and a total area of 12.3×12.3 mm, and alens with an image demagnification factor of 1.45×. For such detectorand lens combination, 4×4 binning is preferred, resulting in asuper-pixel size of approximately 100×100 microns, which translates toapproximately 150 micron resolution in the object plane at the ECLelectrode. Particularly advantageous, for their low cost and size, isthe use of non-cooled cameras or cameras with minimal cooling(preferably to about −20° C., about −10° C., about 0° C., or highertemperatures). In a preferred embodiment, the light detection subsystemincludes a lens assembly consisting of a series of lens elements (904and 905) designed to produce a telecentric view of the imaged wells andan optical bandpass filter (903) in the optical path within the lensassembly such that the light rays passing through the filter are atsubstantially normal incidence with respect to the filter. In theembodiment illustrated in FIG. 9, the camera is provided a telecentricview of the imaged wells (901).

The housing top of the plate handling system further includes a platestacker mounted on the housing top, above the plate introductionapertures, wherein the plate stackers are configured to receive ordeliver plates to the plate elevators. The plate stacker can include aremovable stacking nest configured to house a plurality of plates andprevent shifting of plates on the instrument, thereby coordinating theproper introduction of each plate in the stacking nest onto the plateelevator. In one embodiment, the stacking nest can accommodate at least5 plates, and preferably at least 10 plates, and the stacking nest canaccommodate a plate nesting extension element configured to furtherextend the capacity of the stacking nest. The plate elevator comprises aplate detection sensor, e.g., a capacitance sensor, and the stacker canalso include a plate detection sensor, e.g., a capacitance, weight, oroptical sensor.

A method is provided for using the apparatus for conducting measurementsin multi-well plates. The plates may be conventional multi-well plates.Measurement techniques that may be used include, but are not limited to,techniques known in the art such as cell culture-based assays, bindingassays (including agglutination tests, immunoassays, nucleic acidhybridization assays, etc.), enzymatic assays, colorometric assays, etc.Other suitable techniques will be readily apparent to one of averageskill in the art.

Methods for measuring the amount of an analyte also include techniquesthat measure analytes through the detection of labels which may beattached directly or indirectly (e.g., through the use of labeledbinding partners of an analyte) to an analyte. Suitable labels includelabels that can be directly visualized (e.g., particles that may be seenvisually and labels that generate an measurable signal such as lightscattering, optical absorbance, fluorescence, chemiluminescence,electrochemiluminescence, radioactivity, magnetic fields, etc). Labelsthat may be used also include enzymes or other chemically reactivespecies that have a chemical activity that leads to a measurable signalsuch as light scattering, absorbance, fluorescence, etc. The formationof product may be detectable, e.g., due a difference, relative to thesubstrate, in a measurable property such as absorbance, fluorescence,chemiluminescence, light scattering, etc. Certain (but not all)measurement methods that may be used with solid phase binding methodsaccording to the invention may benefit from or require a wash step toremove unbound components (e.g., labels) from the solid phase

In one embodiment, a measurement done with the apparatus of theinvention may employ electrochemiluminescence-based assay formats, e.g.electrochemiluminescence based immunoassays. The high sensitivity, broaddynamic range and selectivity of ECL are important factors for medicaldiagnostics. Commercially available ECL instruments have demonstratedexceptional performance and they have become widely used for reasonsincluding their excellent sensitivity, dynamic range, precision, andtolerance of complex sample matrices. Species that can be induced toemit ECL (ECL-active species) have been used as ECL labels, e.g., (i)organometallic compounds where the metal is from, for example, the noblemetals of group VIII, including Ru-containing and Os-containingorganometallic compounds such as the tris-bipyridyl-ruthenium (RuBpy)moiety, and (ii) luminol and related compounds. Species that participatewith the ECL label in the ECL process are referred to herein as ECLcoreactants. Commonly used coreactants include tertiary amines (e.g.,see U.S. Pat. No. 5,846,485), oxalate, and persulfate for ECL from RuBpyand hydrogen peroxide for ECL from luminol (see, e.g., U.S. Pat. No.5,240,863). The light generated by ECL labels can be used as a reportersignal in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808,herein incorporated by reference). For instance, an ECL label can becovalently coupled to a binding agent such as an antibody, nucleic acidprobe, receptor or ligand; the participation of the binding reagent in abinding interaction can be monitored by measuring ECL emitted from theECL label. Alternatively, the ECL signal from an ECL-active compound maybe indicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants). For more background on ECL, ECL labels,ECL assays and instrumentation for conducting ECL assays see U.S. Pat.Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623;5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141;5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863;6,207,369; 6,214,552 and 5,589,136 and Published PCT Nos. WO99/63347;WO00/03233; WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931and WO98/57154, all of which are incorporated herein by reference.

In certain embodiments, plates adapted for use inelectrochemiluminescence (ECL) assays are employed as described in U.S.Pat. No. 7,842,246. The apparatus of the invention can use plates thatare configured to detect ECL from one well at a time or more than onewell at a time. As described above, plates configured to detect ECL onewell at a time or more than one well at a time include electrode andelectrode contacts that are specifically patterned to allow applicationof electrical energy to electrodes in only one well at a time or morethan one well at a time. The apparatus may be particularly well-suitedfor carrying out assays in plates containing dry reagents and/or sealedwells, e.g., as described in U.S. application Ser. No. 11/642,970 ofGlezer et al.

In one embodiment, the method comprises: (a) introducing a plate to aplate stacker, (b) opening the light-tight door, (c) lowering the platefrom the plate stacker to the lifting platform on the plate translationstage, (d) sealing the light-tight door, (e) translating the plate toposition one or more wells under the light detector, (f) detectingluminescence from the one or more wells, (g) opening the light-tightdoor, (h) translating the plate to a position under a plate stacker, and(i) raising the plate to the plate stacker. In a preferred embodiment,the method also includes reading a plate identifier on the plate andidentifying the plate configuration, translating the plate to positionthe one or more wells under the light detector, optionally imaging oneor more alignment features on the contact mechanism and adjusting theposition of the light detector relative to the contact mechanism, andselectively applying potential within one or more interrogation zonesbased on the plate configuration. The method may further comprisetranslating the plate carriage to position one or more additional wellsunder the light detector and detecting luminescence from the one or moreadditional wells. The method may also, optionally, comprise applyingelectrical energy to electrodes in one or more of the wells (e.g., toinduce electrochemiluminescence).

ECL-based multiplexed testing is described in U.S. Publications2004/0022677 and 2004/0052646 of U.S. application Ser. Nos. 10/185,274and 10/185,363, respectively; U.S. Publication 2003/0207290 of U.S.application Ser. No. 10/238,960; U.S. Publication 2003/0113713 of U.S.application Ser. No. 10/238,391; U.S. Publication 2004/0189311 of U.S.application Ser. No. 10/744,726; and U.S. Publication 2005/0142033 ofU.S. application Ser. No. 10/980,198.

A method is also provided for conducting assays for biological agentsusing the apparatus described herein. In one embodiment, the method is abinding assay. In another embodiment, the method is a solid-phasebinding assay (in one example, a solid phase immunoassay) and comprisescontacting an assay composition with one or more binding surfaces thatbind analytes of interest (or their binding competitors) present in theassay composition. The method may also include contacting the assaycomposition with one or more detection reagents capable of specificallybinding with the analytes of interest. The multiplexed binding assaymethods according to preferred embodiments can involve a number offormats available in the art. Suitable assay methods include sandwich orcompetitive binding assays format. Examples of sandwich immunoassays aredescribed in U.S. Pat. Nos. 4,168,146 and 4,366,241. Examples ofcompetitive immunoassays include those disclosed in U.S. Pat. Nos.4,235,601; 4,442,204; and 5,208,535 to Buechler et al. In one example,small molecule toxins such as marine and fungal toxins can beadvantageously measured in competitive immunoassay formats.

Binding reagents that can be used as detection reagents, the bindingcomponents of binding surfaces and/or bridging reagents include, but arenot limited to, antibodies, receptors, ligands, haptens, antigens,epitopes, mimitopes, aptamers, hybridization partners, andintercalaters. Suitable binding reagent compositions include, but arenot limited to, proteins, nucleic acids, drugs, steroids, hormones,lipids, polysaccharides, and combinations thereof. The term “antibody”includes intact antibody molecules (including hybrid antibodiesassembled by in vitro re-association of antibody subunits), antibodyfragments, and recombinant protein constructs comprising an antigenbinding domain of an antibody (as described, e.g., in Porter & Weir, J.Cell Physiol., 67 (Suppl 1):51-64, 1966; Hochman et al., Biochemistry12:1130-11.35, 1973; hereby incorporated by reference). The term alsoincludes intact antibody molecules, antibody fragments, and antibodyconstructs that have been chemically modified, e.g., by the introductionof a label.

Measured, as used herein, is understood to encompass quantitative andqualitative measurement, and encompasses measurements carried out for avariety of purposes including, but not limited to, detecting thepresence of an analyte, quantitating the amount of an analyte,identifying a known analyte, and/or determining the identity of anunknown analyte in a sample. According to one embodiment, the amountsthe first binding reagent and the second binding reagent bound to one ormore binding surfaces may be presented as a concentration value of theanalytes in a sample, i.e., the amount of each analyte per volume ofsample.

Analytes may be detected using electrochemiluminescence-based assayformats. Electrochemiluminescence measurements are preferably carriedout using binding reagents immobilized or otherwise collected on anelectrode surface. Especially preferred electrodes includescreen-printed carbon ink electrodes which may be patterned on thebottom of specially designed cartridges and/or multi-well plates (e.g.,24-, 96-, 384- etc. well plates). Electrochemiluminescence from ECLlabels on the surface of the carbon electrodes is induced and measuredusing an imaging plate reader as described in copending U.S. applicationSer. Nos. 10/185,274 and 10/485,363 (both entitled “Assay Plates, ReaderSystems and Methods for Luminescence Test Measurements”, filed on Jun.28, 2002, hereby incorporated by reference). Analogous plates and platereaders are now commercially available (MULTI-SPOT® and MULTI-ARRAY®plates and SECTOR® instruments, Meso Scale Discovery, a division of MesoScale Diagnostics, LLC, Rockville, Md.).

In one embodiment, antibodies that are immobilized on the electrodeswithin the plates may be used to detect the selected biological agent ina sandwich immunoassay format. In another embodiment, microarrays ofantibodies, patterned on integrated electrodes within the plates, willbe used to detect the plurality of the selected biological agents in asandwich immunoassay format. Accordingly, each well contains one or morecapture antibodies immobilized on the working electrode of the plateand, optionally, in dry form or as separate components, e.g., in a kit,labeled detection antibodies and all additional reagents necessary foranalysis of samples, and for carrying out positive and negativecontrols.

Patents, patent applications, publications, and test methods cited inthis disclosure are incorporated herein by reference in their entirety.The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe claims.

PARTS LIST Reference No. Part Name 100 Apparatus 110 Light detectionsystem with light detectors 120 Plate handling system 130 Light tightenclosure 231 Housing 232 Housing top 233 Housing bottom 234 Housingfront 235 Housing rear 236, 237 Plate introduction/ejection 238 Bar codereader 240 Removable drawer 400 Plate elevator mechanism 401, 402 Platelifting platform 403 Plate translation stage 404 Plate carriage withopening 405, 406, 407 Alignment pins 408, 409, 410 Alightment holes 411Spring loaded pin 412 Hole in plate carriage 404 413 Electrical contactmechanism on housing tip 232 414 Companion electrical contact mechanismon drawer 415 X-Y Frame 416, 417 Alignment latches 418, 419 Alignmentcatches 420 Opening in carriage 422, 424 Rails 426 Multi-well plate 501First stop 502 Plate clamp arm 503 Bracket 504 Leg 506 Leg 507 Ramp 508Ramp 509 First latch member 510 Actuating rod 510a Extended portion ofactuating rod 511 Pedal 512 Spring 513 Second Stop 514 Spring 515 Biasedclamp 515a Extended portion of biased clamp 515b Biased end of biasedclamp 516 Ejector 522 Skirt 524 Fulcrum 526 Sheath 528, 530 Ends of arm502 531 Pivot point 532 Spring for biased clamp 515 534 Over-travelpreventer 536 Conductive bottom surface 701 Platform 702, 703, 704, 705Interrogation zones on platform 701 706, 707, 708, 709, Workingelectrodes on platform 701 710, 711, 712, 713 714, 715, 716, 717,Counter electrodes on platform 701 718, 719, 720, 721 722 Aligning lightoutlet 723 Shaft 724 Gear mechanism 725, 726, 727, 728 Light outlets 729Microprocessor 730 Power source 731 DAC 732 Low-pass filter 733 Low-passfilter 734 Current monitor 736 ADC 737 Power source 738 Multiplexer 739LED 740 Bottom counter electrode 742, 744 Bottom working electrodes 750Well working electrode 752, 754 Well counter electrodes 760 Bottomworking electrode 762 Bottom counter electrode 801 Light detectorhousing 802 Cast component 803 Buckle or clamp 804 Screw 805 Gear 901Imaged well 902 Camera 903 Optical bandpass filter 904 Lens 905 Lens

We claim:
 1. An instrument comprising: a contact platform, wherein thecontact platform comprises at least a pair of electrical contacts,wherein the at least a pair of electrical contacts comprise upstanding,spring-loaded pins, a controller operatively connected to a voltagesource to conduct a voltage to the at least a pair of electricalcontacts, a plate carriage frame adapted to transport an addressablemulti-well plate and to position the multi-well plate relative to thecontact platform, such that the voltage can be applied to one or morewells on the multi-well plate, and an optical sensor positioned abovethe contact platform and a first alignment mechanism comprising a lightsource projecting from the contact platform toward the optical sensor toalign the contact platform relative to the optical sensor.
 2. Theinstrument of claim 1, further comprising a second alignment mechanismcomprising a plurality of apertures located on the plate carriage frameand wherein the light source projecting from the platform is illuminatedthrough the apertures to further align the plate carriage frame with thecontact platform.
 3. The instrument of claim 2, wherein the platecarriage frame comprises a rectangular opening sized and dimensioned tosupport a skirt on a perimeter of the multi-well plate.
 4. Theinstrument of claim 3, wherein the plurality of apertures are positionedon at least two sides of the rectangular opening.
 5. The instrument ofclaim 1, wherein the plate carriage frame comprises a latching mechanismto retain the multi-well plate to the plate carriage frame.
 6. Theinstrument of claim 1 further comprising a focusing mechanism disposedon the plate carriage frame allowing the optical sensor to be focusedrelative to the focusing mechanism.
 7. The instrument of claim 1 furthercomprising a third alignment mechanism comprising an electricallyconductive surface located on the plate carriage frame such that whenthe at least a pair of electrical contacts on the contact platform arebrought in contact with the electrically conductive surface electricalcurrent flows among the electrical contacts on the contact platform toindicate a predetermined distance between the electrical contacts andthe plate carriage frame.
 8. An instrument for conducting luminescenceassays in a multi-well plate, the instrument comprising a plate handlingsubsystem including a plate carriage for supporting the multi-wellplate, wherein the plate carriage comprises a frame and a plate latchingmechanism comprising: (a) a plate carriage ledge; (b) a plate clamp armperpendicular to the plate carriage ledge and comprising a proximate endand a distal end relative to the plate carriage ledge, wherein the plateclamp arm is attached to the frame at the proximate end and the plateclamp arm is rotatable in an x-y plane at the distal end, and the plateclamp arm further comprises an upper clamp including an angled surfaceconfigured to engage with the multi-well plate; (c) a plate positioningelement comprising a rod, a pedal and a spring, wherein the rod isperpendicular to the plate clamp arm, parallel to the plate carriageledge, and attached to the distal end of the plate clamp arm via thespring, and the pedal is attached to the rod at an angle; and (d) aplate wall parallel to the plate clamp arm and perpendicular to anddisposed between the plate positioning element and the plate carriageledge, the plate wall comprising (i) a lower plate clamp configured toengage with a multi-well plate skirt, and (ii) a lower plate clamp rampconfigured to drive the lower plate clamp toward the multi-well plateskirt.
 9. The instrument of claim 8 wherein the plate wall furthercomprises a plate ejector element configured to disengage the lowerplate clamp from the skirt.
 10. An instrument for conductingluminescence assays in a multi-well plate, the instrument comprising aplate handling subsystem including a plate carriage for supporting themulti-well plate, and a plate latching mechanism, wherein the multi-wellplate has at least a first side, a second side, a third side and afourth side and wherein the first side and the third side aresubstantially parallel to each other and the second side and the fourthside are substantially parallel to each other, wherein the platecarriage defines an aperture having a shape substantially the same asthe multi-well plate and having dimensions smaller than the multi-wellplate to support a ledge positioned around a perimeter of the multi-wellplate, wherein the plate carriage further comprises a first stop and asecond stop corresponding to the first side and the second side of themulti-well plate, respectively, wherein the plate latching mechanism ismovable from an open configuration to accept one multi-well plate to aclamping position to latch the multi-well plate to the plate carriage,wherein the plate latching mechanism comprises a first latching memberbiased to the clamping position and having a pedal adapted to push thefirst side of the multi-well plate toward the first stop and a plateclamp arm biased to the clamping position and having a bracket pivotallyconnected to the plate clamp arm and is adapted to push the second sidetoward the second stop, wherein the plate latching mechanism isconnected to the plate clamp arm, and wherein the plate latchingmechanism comprises at least one biased clamp positioned proximate tothe second stop to clamp to a skirt of a multi-well tray to the platecarriage.
 11. The instrument of claim 10, wherein the bracket comprisesat least two legs and both are in contact with the fourth side of themulti-well tray.
 12. The instrument of claim 11, wherein at least oneleg comprises a ramp to apply force orthogonal to a plane of themulti-well tray.
 13. The instrument of claim 10, wherein the firstlatching member comprises an actuating rod, which is biased to theclamping position by a spring.
 14. The instrument of claim 13, whereinthe pedal is attached to the actuating rod, and the plate carriage has athird stop to force the pedal to move toward and away from themulti-well tray as the actuating rod is moved.
 15. The instrument ofclaim 13, wherein the pedal and plate clamp arm retract in the openconfiguration.
 16. The instrument of claim 10 further comprises anejector that moves the multi-well tray away from the first stop or thesecond stop.
 17. The instrument of claim 16, wherein the ejector isspring-loaded.
 18. The instrument of claim 16, wherein the ejectorcomprises an over-travel preventer.